The standardization and clinical validation of the measurement of β–amyloid. (Aβ42) in cerebrospinal fluid (CSF), plasma and urine is described using a commercially available sandwich–type ELISA with 2 IF12 and 3D6 as monoclonal antibodies. The INNOTEST™ β–amyloid(1–42) allows the specific and reliable measurement of (1–42) amyloid peptides in CSF and plasma. The Aβ42 concentrations in serum and urine were below the detection limit. In plasma, no differences were found in Aβ42 levels between controls and patients with different neurodegenerative disorders (Alzheimer's disease (AD), Lewy body disease (LED), others). In contrast, CSF–Aβ42 concentrations were lower in AD and LBD patients as compared to controls. No correlation was found in AD patients between CSF and plasma concentrations of Aβ42 or between CSF Aβ42 levels and blood–brain–barrier function. The quantitative outcome of the test is in part dependent on confounding factors such as tube type, freeze/thaw cycles, temperature of incubation, standard preparation protocol, and antibody selection. Notwithstanding these aspects, it emerged that Aβ42 is a useful biochemical marker for the diagnosis of AD patients, but there is a need for an international Aβ standard, a universally accepted protocol for CSF preparation, and a thorough evaluation of assay performance in function of the boundary conditions.